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Sample GSM4752173

Query DataSets for GSM4752173

Status

Public on Aug 29, 2020

Title

Cultured cortical neurons, Z1 (RNA-seq)

Sample type

SRA

Source name

Cultured cortical neuron

Organism

Homo sapiens

Characteristics

cell type: neurons differentiated from 1st passage of neuroprogenitors from fetal frontal cortex
developmental stage: 21 week of gestational age
karyotype: 46, XX
treatment: 1st passage neuroprogenitors infected with lentivirus carrying ZsGreen gene and cultured for 4 days
genotype: CON

Treatment protocol

1st passage of human neuroprogenitors were infected with letivirus carrying ZsGreen or DNMT3L gene

Growth protocol

Lentivirus Infected cells were cultured in Neurobasal+2%B27 for 4 days

Extracted molecule

total RNA

Extraction protocol

The total RNA of each sample was extracted from the cells by using TRIzol reagent (Life Technologies, USA)/RNeasy Mini Kit (Qiagen, USA) following the manufacturer’s protocol. Total RNA of each sample was quantified and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.) and 1% agrose gel. 1 μg total RNA with RIN value above 6.5 was used for following library preparation.
Next generation sequencing library preparations were constructed according to the manufacturer’s protocol. The poly(A) mRNA isolation was performed using Poly(A) mRNA Magnetic Isolation Module or rRNA removal Kit. The mRNA fragmentation and priming was performed using First Strand Synthesis Reaction Buffer and Random Primers. First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase and the second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix. The purified double- stranded cDNA by beads was then treated with End Prep Enzyme Mix to repair both ends and add a dA- tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor- ligated DNA was then performed using beads, and fragments of ~420 bp (with the approximate insert size of 300 bp) were recovered. Each sample was then amplified by PCR for 13 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with flow cell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned up using beads, validated using an Qsep100 (Bioptic,Taiwan, China), and quantified by Qubit3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 3000

Data processing

Bcl2fastq (v2.17.1.14) software was used for base-calling
To remove technical sequences, including adapters, polymerase chain reaction (PCR) primers, or fragments thereof, and quality of bases lower than 20, pass filter data of fastq format were processed by Cutadapt (V1.9.1) to be high quality clean data.
Reference genome sequences and gene model annotation files of relative species were downloaded from genome website, such as UCSC, NCBI, ENSEMBL. Hisat2 (v2.0.1) was used to index reference genome sequence. Finally, clean data were aligned to reference genome via software Hisat2 (v2.0.1).
In the beginning transcripts in fasta format were converted from known gff annotation file and indexed properly. Then, with the file as a reference gene file, HTSeq (v0.6.1) estimated gene and isoform expression levels from the pair-end clean data.
QC,Cutadapt (V1.9.1); QC assessment, FastQC (V0.10.1); Alignment, HISAT2 (v2.0.1); Gene Expression, HTSEQ (v0.6.1); Assembly, Stringtie (V1.3.3b); Different expression gene analysis, DESeq2(V1.6.3), DESeq (v1.18.0), EdgeR (V3.4.6), Cuffdiff (v2.2.1)
Genome_build: GRCh37.91/hg19
Supplementary_files_format_and_content: xls file includes FPKM values for each sample

Submission date

Aug 28, 2020

Last update date

Aug 29, 2020

Contact name

JIE LU

E-mail(s)

[email protected]

Phone

18900910753

Organization name

China Medical University